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Effect of prenatal ethanol exposure during the brain growth spurt of the guinea pig
Neurotoxicology and Teratology 23 (2001) 355 – 364
Effect of prenatal ethanol exposure during the brain growth spurt of the guinea pig M.L. Byrnesa, J.N. Reynoldsa,b, J.F. Briena,* a
Department of Pharmacology and Toxicology, Faculty of Health Sciences, Queen’s University, Kingston, Ontario, Canada K7L 3N6 b Department of Anaesthesiology, Faculty of Health Sciences, Queen’s University, Kingston, Ontario, Canada K7L 3N6 Received 18 October 2000; received in revised form 15 March 2001; accepted 9 April 2001
Abstract This study tested the hypothesis that prenatal ethanol exposure during the last third of gestation, including the brain growth spurt (BGS), in the guinea pig produces neurobehavioural teratogenicity, manifesting as brain growth restriction and hyperactivity. Pregnant guinea pigs (term, about gestational day (GD) 68) received oral administration of ethanol (2 g/kg maternal body weight per day on GD 43 and/or GD 44 and then 4 g/kg maternal body weight per day from GD 45 to GD 62), isocaloric-sucrose/pair-feeding, or water. Maternal blood ethanol concentration (BEC) on GD 57 or 58, at 1 h after the daily dose, was 340 ± 76 mg/dl (n = 8). Ethanol treatment decreased brain, cerebral cortical, hippocampal, and cerebellar weights at GD 63 ( P < 0.05), and decreased brain and cerebral cortical weights at postnatal day 10 ( P < 0.05), with no effect on body weight and no apparent effect on spontaneous locomotor activity. The data demonstrate that, in the guinea pig, prenatal ethanol exposure during the last third of gestation, including the BGS, decreases brain weight that persists into postnatal life, which is associated with growth restriction of the cerebral cortex. However, this prenatal ethanol exposure regimen, including the BGS, does not increase spontaneous locomotor activity in contrast to the persistent hyperactivity that occurs after chronic ethanol exposure throughout gestation. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Prenatal ethanol exposure; Brain growth spurt; Cerebral cortex; Hippocampus; Cerebellum; Spontaneous locomotor activity; Guinea pig
1. Introduction Consumption of ethanol during pregnancy can produce a broad spectrum of toxic effects on the developing fetus, including teratogenicity, spontaneous abortion, and stillbirth [3]. The teratogenic effects of ethanol in the human can present as the fetal alcohol syndrome (FAS) [14]. The most debilitating and permanent consequence of ethanol teratogenesis is the central nervous system (CNS) dysfunction, which can manifest as intellectual, neurological, and behavioural abnormalities. Experimental animal studies have demonstrated that the cerebral cortex, hippocampus, and cerebellum are among the brain target sites of ethanol CNS teratogenesis [24,34]. Considerable advances have been made in the last decade in our understanding of FAS, but
* Corresponding author. Tel.: +1-613-533-6114; fax: +1-613-5336412. E-mail address: [email protected] (J.F. Brien).
the mechanism(s) of ethanol CNS teratogenesis remain(s) to be elucidated [4,34]. The development of the CNS in the human and other mammalian species can be divided into distinct stages, but with considerable overlap of the ontogenic events that occur [24]. Exposure to ethanol during different stages of gestation can produce different deleterious intellectual, neurological, and/or behavioural outcomes, thereby reflecting temporal vulnerability during development. The developing brain may be especially sensitive to the teratogenic effects of ethanol during the brain growth spurt (BGS), which is characterized by a rapid increase in brain weight, proliferation of astroglial and oligodendroglial cells, neuronal axonal elongation, dendritic arborization, and synaptogenesis [24]. In the human, the BGS is a perinatal event that begins in the third trimester and continues into early postnatal life [19]. It has been suggested that cessation of maternal consumption of ethanol before the beginning of the third trimester can limit the severity or prevent the occurrence of some of ethanol’s CNS teratogenic effects [16,17].
0892-0362/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 2 - 0 3 6 2 ( 0 1 ) 0 0 1 5 0 - 7
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Another important factor influencing the CNS teratogenic effects of ethanol for exposure during distinct periods of development is the fact that not all brain regions develop at the same time, and thus, depending on the developmental stage during which ethanol exposure occurs, some brain regions may be more affected than others [24]. In addition, within a given brain region, different neuronal cell populations have different temporal patterns of development that may influence their relative vulnerability to the teratogenic effects of ethanol [24]. When using animal models to study temporal vulnerability to ethanol exposure, one must take into account the timing of the various stages of brain development, such as the BGS, in relation to birth. In the rat, the BGS occurs primarily after birth, with the majority of the BGS occurring during the first 2 weeks of postnatal life [35]. Thus, exposure to ethanol during the BGS in the rat requires administration of ethanol to the neonate. The objective of this study was to characterize, in the guinea pig, ethanol CNS teratogenicity for prenatal ethanol exposure during the last third of gestation, including the BGS. The following hypothesis was tested: prenatal ethanol exposure during the last third of gestation, including the BGS, in the guinea pig produces neurobehavioural teratogenicity, manifesting as brain growth restriction and hyperactivity. The guinea pig was selected as the experimental animal because of its extensive prenatal brain development including the BGS, the majority of which occurs between gestational day (GD) 45 and 55 (term, about GD 68) [18]. Therefore, in the guinea pig, maternal administration of ethanol during the last third of gestation results in offspring being exposed to ethanol in utero during the BGS, an apparent critical period of vulnerability. People with FAS have been exposed to ethanol during prenatal life, as a consequence of maternal ingestion of ethanol. Thus, administration of ethanol to the pregnant guinea pig would appear to model more closely the human situation, than is the case for the rat.
2. Methods 2.1. Experimental animals Nulliparous female Dunkin – Hartley strain guinea pigs (500 –600 g body weight) were bred with male Dunkin – Hartley strain guinea pigs using an established procedure [22]. GD 0 was defined as the last day of full vaginalmembrane opening. On GD 1, pregnant animals were housed individually in cages in a room with a 12-h light/ 12-h dark cycle with lights on at 0700 h and ambient room temperature of 23C. Vaginal-membrane status and general health of the pregnant animals were monitored, and body weight was measured daily throughout gestation. All guinea pigs were cared for according to the principles and guidelines of the Canadian Council on Animal Care. The experi-
mental protocol was approved by the Queen’s University Animal Care Committee. 2.2. Animal treatment regimens and maternal blood ethanol concentration Two investigations were conducted, in which offspring were studied prenatally at GD 63 or postnatally at PD 10. In the GD 63 study, six pregnant guinea pigs were studied for each of the ethanol, isocaloric-sucrose/pair-fed and water treatment groups; in the PD 10 study, four pregnant guinea pigs were studied for each treatment group. A preliminary study demonstrated that oral administration of 4 g ethanol/kg maternal body weight per day starting on GD 45 resulted in maternal death in 5 of 11 pregnant guinea pigs. In view of this 45% incidence of maternal lethality, the ethanol dosage regimen was modified to provide maternal exposure to a lower daily dose of ethanol (2 g ethanol/kg maternal body weight per day) starting on GD 43, for the first 2 days of treatment in order to allow the pregnant animal to develop tolerance to the maternal toxicity of the 4 g ethanol/kg maternal body weight per day regimen, which began on GD 45. Each pregnant animal was randomly assigned to one of the following oral treatment regimens. The ethanol regimen was administered as 2 g ethanol/kg maternal body weight per day on GD 43 and/or 44 and then as 4 g ethanol/kg maternal body weight per day from GD 45 to 62 in the form of an aqueous ethanol solution (30% v/v in tap water) with ad libitum access to pellet food (Purina Guinea Pig Chow 50251) and water. The 4 g ethanol/kg maternal body weight per day regimen was selected for study because it has been shown to produce persistent neurobehavioural changes in offspring, with minimal embryonic/fetal or maternal lethality, when given throughout gestation [2,12]. For the isocaloric-sucrose/pair-feeding regimen, each pregnant animal was paired to an ethanoltreated guinea pig, received sucrose (42% w/v in tap water, isocaloric and isovolumetric to the daily dose of ethanol) and was given food in an amount equivalent to that consumed by the ethanol-treated animal, with free access to water. Virtually all the food provided on each day of the treatment period was consumed by the pregnant animal. For the water regimen, water (isovolumetric to the daily dose of ethanol) was administered with ad libitum access to food and water. Each animal received the daily treatment by oral intubation into the mouth as two equally divided doses 2 h apart, with the first dose given between 1000 and 1200 h and the second dose given 2 h later. Each animal was treated from GD 43 to 62 (term, about GD 68) to include the BGS. Thereafter, each pregnant animal had ad libitum access to food and water. On GD 57 or 58, maternal blood was taken from an ear blood vessel at 1 h after the second divided dose of ethanol, isocaloric-sucrose, or water for the determination of ethanol concentration by an established gas-liquid
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chromatographic procedure [32]. The 200-ml blood sample was collected from each pregnant guinea pig in the study over a maximum time period of 5 min, in which the animal was restrained only during the blood vessel puncture. The time of blood sampling was selected in view of a previous pharmacokinetic study [13] of ethanol disposition in the maternal-fetal unit, in which the apparent maximum blood ethanol concentration (BEC) occurred at 1 h after the last divided dose of 4 g ethanol/kg maternal body weight. Blood sampling was performed on GD 57 or 58 in order to compare the ethanol concentration data with those determined in previous studies, in which ethanol was administered throughout gestation. 2.3. Pregnancy outcome At birth (PD 0), each maternal guinea pig and its litter were moved from the cage housing, used up to and including parturition, to a large plastic bin with wood chip bedding. The litter size and number of live offspring were recorded, and the presence of gross dysmorphology was assessed. On PD 1, the body weight and gender of the offspring of each litter were determined. The offspring were monitored daily for body weight and general health. Perinatal death was defined as death that occurred at parturition or during the first 10 days of postnatal life. 2.4. Measurement of spontaneous locomotor activity On PD 10, the cumulative spontaneous locomotor activity of the individual offspring from each litter was measured in an open-field apparatus using an Opto-Varimex1 monitor (Columbus Instruments; Columbus, OH). The apparatus (42 42 21 cm high) was equipped with blackened walls, a clear plastic top, and two arrays of infrared beams that were set at 3 cm above the floor to measure horizontal spontaneous locomotor activity (ambulatory, exploratory movement) and at 8 cm above the floor to measure vertical spontaneous locomotor activity (climbing, rearing). The cumulative number of infrared-beam breaks in each of the horizontal and vertical planes were recorded at 10-min intervals for a 60-min period. The testing was performed in a room with ambient temperature of 23C and standard fluorescent lighting, in which background noise was minimized. 2.5. Offspring body, brain, and brain region weights On GD 63, individual pregnant guinea pigs from each of the three treatment groups were euthanized by halothane anesthesia followed by decapitation. The litter of each pregnant animal was delivered by cesarean section, and each fetus was decapitated. On PD 10, randomly selected offspring from each litter were weighed, and euthanized by halothane anesthesia followed by decapitation. The offspring’s brain was excised and weighed. The
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hippocampus, cerebral cortex, and cerebellum were dissected and weighed. 2.6. Data analysis The data are presented as group means ± S.E.M. of the average littermate values for the individual litters. There was no heterogeneity of variance for the data of the three treatment groups, as determined by Bartlett’s test. Therefore, parametric statistical analysis was conducted using analysis of variance (ANOVA). For the weight data, oneway, randomized-design ANOVA was conducted. If a treatment effect was indicated by a statistically significant F statistic ( P < 0.05), then Newman-Keuls post hoc test was conducted to determine which treatment groups were statistically different. Spontaneous locomotor activity data were analyzed using two-way ANOVA with respect to treatment and time.
3. Results 3.1. Maternal BEC, food intake, and body weight The maternal BEC of the pregnant guinea pigs in the ethanol treatment group at 1 h after the second divided dose on GD 57 or 58 was 340 ± 76 mg/dl (n = 8). There was no measurable ethanol in the blood of the pregnant guinea pigs that received isocaloric-sucrose/pair-feeding or water treatment. The average daily food intake and maternal body weight data of the pregnant guinea pigs in the three experimental groups for the GD 43 to 62 treatment period are presented in Fig. 1 for the study involving GD 63 offspring and in Fig. 2 for the study involving PD 10 offspring. For each study, the average daily food intake of the pregnant animals was decreased in the ethanol treatment group compared with the water treatment group ( P < 0.05). However, the maternal body weight during and after the treatment period was not different among the ethanol, isocaloric-sucrose/pair-fed and water treatment groups. 3.2. Pregnancy outcome The pregnancy outcome data for the three treatment groups of each of the two studies at GD 63 and PD 10 are presented in Tables 1 and 2, respectively. There was no maternal death for the ethanol, isocaloric-sucrose/pairfed, and water treatment groups of the two studies. There was one incident of spontaneous abortion in the isocaloric-sucrose/pair-fed pregnant guinea pigs of the study at GD 63, and two incidents of spontaneous abortion in the ethanol-treated pregnant guinea pigs of the study at PD 10. There was no perinatal death for the three treatment groups of the two studies. There was no difference in litter size, or percentage of male and female littermates
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neous locomotor activity during the 60-min period ( P < 0.05). 3.4. Offspring body, brain, and brain region weights There was no gender difference in the offspring body, brain, cerebral cortical, hippocampal, or cerebellar weight data for any of the three treatment groups at GD 63 or PD 10 (data not shown). Consequently, the data of the male and female offspring in each treatment group were combined. The body, brain, cerebral cortical, hippocampal, and cerebellar weight data for GD 63 offspring of the three treatment groups are presented in Fig. 4. There was no difference in body weight among the three treatment groups. Brain, cerebral cortical, hippocampal, and cerebellar weights were decreased by 17.3%, 19.7%, 13.6%, and 13.4%, respectively, for the ethanol group compared with the isocaloric-sucrose/pair-fed control group ( P < 0.05), and were decreased by 21.1%, 21.1%, 23.3%, and 20.9%,
Fig. 1. Average daily food intake during treatment and maternal body weight of pregnant guinea pigs that received oral administration of ethanol (n = 6), isocaloric-sucrose/pair-feeding (n = 6) or water (n = 6) during the last third of gestation, including the BGS (GD 43 to GD 62; term, about GD 68), for the GD 63 study. The data are presented as group means ± S.E.M. Treatment period is indicated by the shaded box. Groups means with different letters are statistically different from each other ( P < 0.05).
among the three treatment groups at GD 63. There was no difference in length of gestation, litter size, average littermate birth weight, or percentage of male and female littermates among the three treatment groups at PD 10. 3.3. Spontaneous locomotor activity There was no gender difference in the spontaneous locomotor activity data of PD 10 offspring for any of the three treatment groups (data not shown), and the data of the male and female offspring in each treatment group were combined. The cumulative horizontal and vertical spontaneous locomotor activity data for PD 10 male and female offspring of the three treatment groups for the 60min observation period are presented in Fig. 3. Based on statistical analysis of the four litters in each treatment group, there was no apparent treatment effect for horizontal or vertical spontaneous locomotor activity among the ethanol, isocaloric-sucrose/pair-fed and water groups, but there was a time effect of cumulative horizontal sponta-
Fig. 2. Average daily food intake during treatment and material body weight of pregnant guinea pigs that received oral administration of ethanol (n = 4), isocaloric-sucrose/pair feeding (n = 4) or water (n = 4) during the last third of gestation, including the BGS (GD 43 to GD 62; term, about GD 68), for the PD 10 study. The data are presented as group means ± S.E.M. Treatment period is indicated by the shaded box. Groups means with different letters are statistically different from each other ( P < 0.05).
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Table 1 Effects of maternal administration of ethanol, isocaloric-sucrose/pair-feeding, or water during the last third of gestation, including the BGS, on pregnant outcome of the guinea pigs whose offspring were studied at GD 63 Pregnancy outcome variable
Ethanol (6)
Treatment sucrose (7)
Water (6)
Maternal death Spontaneous abortion Perinatal death Litter size Male littermates (%) Female littermates (%)
0 0 0 3.7 ± 0.8 (6) 49.7 ± 17.7 (6) 50.3 ± 17.7 (6)
0 1 0 3.8 ± 0.9 (6) 62.2 ± 26.5 (6) 37.8 ± 26.5 (6)
0 0 0 3.3 ± 0.5 (6) 54.2 ± 18.1 (6) 45.8 ± 18.1 (6)
The number of pregnant guinea pigs or litters is reported in parentheses. The data for maternal death, spontaneous abortion, and perinatal death are reported as the number of occurrences. The other data are reported as group means ± S.E.M. of average littermate values for the individual litters.
respectively, for the ethanol group compared with the water control group ( P < 0.05). However, there was no difference in the brain region weight/brain weight ratio data among the ethanol, isocaloric-sucrose/pair-fed and water treatment groups, respectively: cerebral cortical weight/brain weight ratio, 0.578 ± 0.008, 0.594 ± 0.008, and 0.577 ± 0.007, respectively; hippocampal weight/ brain weight ratio, 0.088 ± 0.005, 0.084 ± 0.002, and 0.091 ± 0.003, respectively; and cerebellar weight/brain weight ratio, 0.107 ± 0.004, 0.103 ± 0.002, and 0.107 ± 0.003, respectively. The body, brain, cerebral cortical, hippocampal, and cerebellar weight data for PD 10 male and female offspring of the three treatment groups are presented in Fig. 5. There was no difference in body, hippocampal, or cerebellar weight among the three treatment groups. Brain and cerebral cortical weights were decreased by 11.6% and 17.1%, respectively, for the ethanol group compared with the isocaloric-sucrose/pair-fed group ( P < 0.05), and were decreased by 15.2% and 16.3%, respectively, for the ethanol group compared with the water control group ( P < 0.05). However, there was no difference in the brain region weight/ brain weight ratio data among the ethanol, isocaloricsucrose/pair-fed, and water treatment groups, respectively; cerebral cortical weight/brain weight ratio, 0.556 ± 0.007, 0.587 ± 0.009, and 0.566 ± 0.010, respectively; hippocampal weight/brain weight ratio, 0.078 ± 0.002, 0.074 ± 0.001,
and 0.072 ± 0.001, respectively; and cerebellar weight/ brain weight ratio, 0.117 ± 0.003, 0.113 ± 0.003, and 0.115 ± 0.003, respectively.
4. Discussion Researchers have been successful in using animal models to investigate the teratogenic effects of ethanol on the developing CNS [21]. In spite of the extensive knowledge of FAS [4,29,34], several important questions regarding the factors that influence the severity of prenatal ethanol exposure-induced CNS damage remain unanswered. One of the most interesting of these questions, from both clinical and experimental points of view, focuses on critical periods during development when the brain may be particularly vulnerable to ethanol exposure, including the BGS. The guinea pig as an experimental animal allows for the examination of both the direct effects (i.e., target fetal site) and indirect effects (i.e., maternal, placental, or other fetal sites) of maternal ethanol administration on fetal brain development during the BGS, which occurs prenatally in this animal species [29]. In the rat, the BGS occurs primarily postnatally [24,28]. Thus, any study attempting to investigate the effects of ethanol on the brain during the BGS in the rat must involve postnatal exposure [28]. Also,
Table 2 Effects of maternal administration of ethanol, isocaloric-sucrose/pair feeding, or water during the last third of gestation, including the BGS, on pregnancy outcome of the guinea pigs whose offspring were studied at PD 10 Pregnancy outcome variable
Ethanol (6)
Treatment sucrose (4)
Water (5)
Maternal death Spontaneous abortion Perinatal death Length of gestation Litter size Average littermate birth weight (g) Male littermates (%) Female littermates (%)
0 2 0 68.3 ± 1.0 (4) 3.4 ± 0.6 (4) 96.2 ± 7.5 (4) 45.8 ± 41.7 (4) 54.2 ± 41.7 (4)
0 1 0 67.8 ± 1.0 (4) 3.0 ± 1.4 (4) 102.9 ± 20.8 (4) 20.8 ± 14.4 (4) 79.2 ± 14.4 (4)
0 0 0 68.6 ± 1.5 (5) 2.4 ± 0.9 (5) 110.7 ± 12.3 (5) 56.7 ± 27.9 (5) 43.3 ± 27.9 (5)
The number of pregnant guinea pigs or litters is reported in parentheses. The data for maternal death, spontaneous abortion, and perinatal death are reported as the number of occurrences. The other data are reported as group means ± S.E.M. of average littermate values for the individual litters.
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Fig. 3. Cumulative horizontal and vertical spontaneous locomotor activity data for a 60-min period of observation of PD 10 offspring of pregnant guinea pigs that received oral administration of ethanol (n = 4 litters), isocaloric-sucrose/pair-feeding (n = 4 litters), or water (n = 4 litters) during the last third of gestation, including the BGS (GD 43 to GD 62; term, about GD 68). The data are presented as group means ± S.E.M. of average littermate values for the individual litters. There was statistically significant effect of time (across the 60-min observation period) for cumulative horizontal activity, F(5,54) = 13.78, P < 0.05.
the potential contribution of ethanol-induced changes in maternal or placental function on offspring brain development cannot be examined in the postnatal rat model [29]. The disposition of ethanol in the maternal-fetal unit in the guinea pig is similar to that for the human situation [8,11,13]. In view of the fact that the neonatal rat has a lower capacity for hepatic biotransformation of ethanol than the adult [31], for a given dose of ethanol normalized to body weight, it would experience a higher BEC for a longer time period than the fetal guinea pig exposed to ethanol in utero, because the predominant route of ethanol
elimination from the maternal-fetal unit is via maternal hepatic biotransformation [8,13]. Thus, in order to clarify that the third trimester is a critical period of brain development for the teratogenic effects of ethanol, more investigation should be conducted using a species that possesses prenatal brain growth characteristics similar to those of the human (e.g., the guinea pig) [33]. In the present study in the guinea pig with maternal ethanol administration restricted to the developmental period from GD 43 to 62, which includes the majority of the BGS, the maternal BEC on GD 57 or 58 at 1 h after
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Fig. 4. Body, brain cerebral cortical, hippocampal, and cerebellar weights for GD 63 offspring of pregnant guinea pigs that received oral administration of ethanol (n = 6 litters), isocaloric-sucrose/pair-feeding (n = 6 litters) during the last third of gestation, including the BGS (GD 43 to GD 62; term, about GD 68). The data are presented as group means ± S.E.M. of average littermate values for the individual litters. Group means with different letters are statistically different from each other ( P < 0.05): brain weight, F(2,15) = 32.09, P < 0.05; cerebral cortical weight, F(2,15) = 17.00, P < 0.05; hippocampal weight, F(2,15) = 11.37, P < 0.05; cerebral weight, F(2,15) = 9.25, P < 0.05.
the daily ethanol dose was about 1.5- to 2-fold higher than the maternal BEC for our previous studies of ethanol teratogenicity, in which the same 4 g ethanol/kg maternal
body weight per day regimen was administered chronically from GD 2 to 67 [1,2,6,9,12,23,25,26]. This difference appears to result from altered pharmacokinetics of ethanol
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Fig. 5. Body, brain, cerebral cortical, hippocampal, and cerebellar weights for PD 10 offspring of pregnant guinea pigs that received oral administration of ethanol (n = 4 litters), isocaloric-sucrose/pair-feeding (n = 4 litters) or water (n = 4 litters) during the last third of gestation, including the BGS (GD 43 to GD 62; term, about GD 68) The data are presented as group means ± S.E.M. of average littermate values for the individual litters. Group means with different letters are statistically different from each other ( P < 0.05): brain weight, F(2,9) = 22.5, P < 0.05; cerebral cortical weight, F(2,9) = 19.98, P < 0.05.
in the maternal-fetal unit due to greater gastrointestinal absorption of ethanol and/or slower elimination of ethanol, via maternal hepatic biotransformation, for the ethanol dosage regimen during the last third of gestation, including
the BGS. It is noteworthy that chronic ethanol ingestion by the adult can induce cytochrome P450 (CYP) 2E1 in the liver and that CYP2E1 catalyzes the hepatic oxidation of ethanol and, hence, the elimination of ethanol at high
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substrate concentration when the alcohol dehydrogenase pathway is saturated [27]. It is reasonable to speculate that the higher maternal BEC in the pregnant guinea pigs that received ethanol only during the last third of gestation, including the BGS, is due to normal rate of hepatic biotransformation of ethanol for this stage of gestation (GD 57 or 58) with no, or minimal, induction of the CYP2E1 metabolic pathway. This is contrast to the situation for chronic ethanol exposure up to this stage of pregnancy (GD 57 or 58) that would induce CYP2E1 and increase maternal hepatic biotransformation of ethanol and the rate of ethanol elimination from the maternal-fetal unit. In the present study, offspring in the ethanol treatment group studied at GD 63 had decreased brain, cerebral cortical, hippocampal, and cerebellar weights compared with the isocaloric-sucrose/pair-fed and water treatment groups. However, offspring in the ethanol treatment group studied at PD 10 only had decreased brain and cerebral cortical weights compared with the isocaloric-sucrose/pairfed and water treatment groups, with no change in hippocampal or cerebellar weight. The data demonstrate that, in the guinea pig, prenatal ethanol exposure during the last third of gestation, including the BGS, decreases brain weight that persists into postnatal life, which is associated with a persistent decrease in cerebral cortical weight. No change was observed in body weight of GD 63 or PD 10 offspring of the ethanol treatment group compared with the isocaloric-sucrose/pair-fed and water treatment groups, indicating that there was selective brain growth restriction. The ethanol-induced decrease in brain and cerebral cortical weights in the young postnatal guinea pig suggests that, although the brain goes through a period of rapid growth during the last third of gestation, each brain region has its own temporal growth spurt and, hence, its own critical period of vulnerability. The data of the present study should not be interpreted to suggest that prenatal ethanol exposure during the last third of gestation, including the BGS, does not alter physiological or biochemical function and/or structure of different neuronal cell types in the hippocampus and cerebellum in postnatal life. Clearly, more research is required before it can be stated that the hippocampus and cerebellum are not vulnerable to the teratogenic effects of prenatal ethanol exposure during the BGS. In view of the fact that the hippocampus of the guinea pig is partially formed by GD 45 (unpublished observation), it is conceivable that the critical period of vulnerability of the hippocampus to prenatal ethanol exposure is earlier in gestation than the general BGS. In addition to microencephaly, hyperactivity is a key behavioural manifestation of FAS in the human and ethanol CNS teratogenicity in experimental animals [21]. Several studies in our laboratory have demonstrated that, in the guinea pig, chronic prenatal ethanol exposure throughout gestation results in a persistent increase in spontaneous locomotor activity and decrease in brain weight in the
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offspring of the ethanol treatment group compared with the isocaloric-sucrose/pair-fed and water treatment groups [2,7,9,23]. In contrast, in our study in which prenatal ethanol exposure was restricted to a time period during the last third of gestation, including the BGS, there was decreased brain weight, but no apparent difference in cumulative horizontal or vertical spontaneous locomotor activity of the offspring at PD 10 among the three treatment groups. Hence, there would appear to be a dissociation between the neurobehavioural deficits produced by chronic ethanol exposure throughout gestation, manifesting as microencephaly and hyperactivity, and the apparent lack of behavioural dysfunction in the presence of microencephaly for ethanol exposure during the BGS. It is conceivable that more sophisticated behavioural assessment, such as the Morris water maze test, which the guinea pig will perform [20], would reveal neurobehavioural dysfunction for ethanol exposure during the BGS. Experiments currently are being conducted to test this concept. Experimental animal studies have shown that there are similarities between the neurobehavioural dysfunction, produced by chronic prenatal ethanol exposure, and by postnatal hippocampal lesioning, such as hyperactivity [30]. The mechanism by which chronic prenatal ethanol exposure produces hyperactivity in the human and experimental animals, including the guinea pig, is not clearly understood. For brain ischemia in the adult gerbil, increased spontaneous locomotor activity is associated with a selective loss of hippocampal CA1 pyramidal cells [5,15]. Similarly, in the young postnatal guinea pig (PD 10) exposed to ethanol prenatally throughout gestation, there is a 30% loss of CA1 pyramidal cells and decreased hippocampal weight, both of which are temporally associated with the hyperactivity [23]. In our study, in which prenatal ethanol exposure was restricted to a time period during the last third of gestation, including the BGS, there was no effect on spontaneous locomotor activity or hippocampal weight, and in a preliminary study, there was no apparent effect on the number of hippocampal CA1 or CA3 pyramidal cells, or dentate gyrus granule cells in PD 10 guinea pig offspring [10]. These findings are consistent with the idea that CA1 pyramidal cell injury as a consequence of prenatal ethanol exposure may be involved in the increased spontaneous locomotor activity. In conclusion, our data demonstrate that, in the guinea pig, prenatal ethanol exposure during the last third of gestation, including the BGS, decreases brain weight that persists into postnatal life and is associated with growth restriction of the cerebral cortex, but does not increase spontaneous locomotor activity. These effects are in contrast to the persistent hyperactivity and decreased brain weight that occur after chronic prenatal ethanol exposure throughout gestation. In order to characterize fully the third trimester, and the BGS in particular, as a critical period of vulnerability for the CNS teratogenic effects of ethanol, more investigation is required using an experimental ani-
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mal, such as the guinea pig, that possesses prenatal and postnatal brain growth characteristics similar to those of the human [33].
Acknowledgments This research was supported by an operating grant (MT8073) from the Canadian Institutes of Health Research. MLB is the recipient of an Eldon Boyd Fellowship, Department of Pharmacology and Toxicology, Queen’s University.
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[16] C.D. Coles, R.T. Brown, I.E. Smith, K.A. Platzman, S. Erikson, A. Falek, Effects of prenatal alcohol exposure at school age: I. Physical and cognitive development, Neurotoxicol. Teratol. 13 (1991) 1 – 11. [17] C.D. Coles, I.E. Smith, P.M. Fernhoff, A. Falek, Neonatal neurobehavioural characteristics as correlates of maternal alcohol use during gestation, Alcohol: Clin. Exp. Res. 9 (1985) 1 – 7. [18] J. Dobbing, J. Sands, Growth and development of the brain and spinal cord of the guinea pig, Brain. Res. 17 (1970) 115 – 123. [19] J. Dobbing, J. Sands, Comparative aspects of the brain growth spurt, Early Hum. Dev. 3 (1979) 79 – 83. [20] H.C. Dringenberg, D.P. Richardson, J.F. Brien, J.N. Reynolds, Spatial learning in the guinea pig: Cued vs. non-cued learning, sex differences, and comparison with rats, Behav. Brain. Res. (in press). [21] C.D. Driscoll, A.P. Streissguth, E.P. Riley, Prenatal ethanol exposure: Comparability of effects in humans and animal models, Neurotoxicol. Teratol. 12 (1990) 231 – 237. [22] H. Elvidge, Production of dated pregnant guinea pigs without postpartum matings, J. Inst. Anim. Tech. 23 (1972) 111 – 117. [23] M.A.S. Gibson, N.S. Butters, J.N. Reynolds, J.F. Brien, Effects of chronic prenatal ethanol exposure on locomotor activity, and hippocampal weight, neurons, and nitric oxide synthase activity of the young postnatal guinea pig, Neurotoxicol. Teratol. 22 (2000) 183 – 192. [24] C. Guerri, Neuroanatomical and neurophysiological mechanisms involved in central nervous system dysfunctions induced by prenatal alcohol exposure, Alcohol: Clin. Exp. Res. 22 (1998) 304 – 312. [25] K.A. Kimura, J. Chiu, J.N. Reynolds, J.F. Brien, Effect of chronic prenatal ethanol exposure on nitric oxide synthase I and III proteins in the hippocampus of the near-term fetal guinea pig, Neurotoxicol. Teratol. 21 (1999) 251 – 259. [26] K.A. Kimura, A.M. Parr, J.F. Brien, Effect of chronic maternal ethanol administration on nitric oxide synthase activity in the hippocampus of the mature fetal guinea pig, Alcohol: Clin. Exp. Res. 20 (1996) 948 – 953. [27] C.S. Lieber, Alcohol and the liver: 1994 update, Gastroenterology 106 (1994) 1085 – 1105. [28] S.E. Maier, W.J.A. Chen, J.A. Miller, J.R. West, Fetal alcohol exposure and temporal vulnerability: Regional differences in alcohol-induced microencephaly as a function of the timing of binge-like exposure during rat brain development, Alcohol: Clin. Exp. Res. 21 (1997) 1418 – 1428. [29] J.D. Reynolds, J.F. Brien, Ethanol neurobehavioural teratogenesis and the role of L-glutamate in the fetal hippocampus, Can. J. Physiol. Pharmacol. 73 (1995) 1209 – 1223. [30] E.P. Riley, S. Barron, J.H. Hannigan, Response inhibition deficits following prenatal alcohol exposure: A comparison to the effects of hippocampal lesions in rats, in: J.R. West (Ed.), Alcohol and Brain Development, Oxford Univ. Press, New York, 1986, pp. 71 – 102. [31] M. Sjoblom, L. Pilstrom, J. Morland, Activity of alcohol dehydrogenase and acetaldehyde dehydrogenase in the liver and placenta during development of the rat, Enzyme 23 (1978) 108 – 115. [32] N.A.E. Steenaart, D.W. Clarke, J.F. Brien, Gas – liquid chromatography analysis of ethanol and acetaldehyde in blood with minimal artifactual acetaldehyde formation, J. Phamacol. Methods 14 (1985) 199 – 212. [33] J.R. West, Fetal alcohol-induced brain damage and the problem of determining temporal vulnerability: A review, Alcohol Drug Res. 7 (1987) 423 – 441. [34] J.R. West, W.J.A. Chen, N.J. Pantazis, Fetal alcohol syndrome: The vulnerability of the developing brain and possible mechanisms of damage, Metab. Brain Dis. 9 (1994) 291 – 322. [35] J.R. West, K.M. Hamre, M.D. Cassell, Effects of ethanol exposure during the third trimester equivalent on neuron number in rat hippocampus and dentate gyrus, Alcohol: Clin. Exp. Res. 10 (1986) 190 – 197.
Effect of prenatal ethanol exposure during the brain growth spurt of the guinea pig M.L. Byrnesa, J.N. Reynoldsa,b, J.F. Briena,* a
Department of Pharmacology and Toxicology, Faculty of Health Sciences, Queen’s University, Kingston, Ontario, Canada K7L 3N6 b Department of Anaesthesiology, Faculty of Health Sciences, Queen’s University, Kingston, Ontario, Canada K7L 3N6 Received 18 October 2000; received in revised form 15 March 2001; accepted 9 April 2001
Abstract This study tested the hypothesis that prenatal ethanol exposure during the last third of gestation, including the brain growth spurt (BGS), in the guinea pig produces neurobehavioural teratogenicity, manifesting as brain growth restriction and hyperactivity. Pregnant guinea pigs (term, about gestational day (GD) 68) received oral administration of ethanol (2 g/kg maternal body weight per day on GD 43 and/or GD 44 and then 4 g/kg maternal body weight per day from GD 45 to GD 62), isocaloric-sucrose/pair-feeding, or water. Maternal blood ethanol concentration (BEC) on GD 57 or 58, at 1 h after the daily dose, was 340 ± 76 mg/dl (n = 8). Ethanol treatment decreased brain, cerebral cortical, hippocampal, and cerebellar weights at GD 63 ( P < 0.05), and decreased brain and cerebral cortical weights at postnatal day 10 ( P < 0.05), with no effect on body weight and no apparent effect on spontaneous locomotor activity. The data demonstrate that, in the guinea pig, prenatal ethanol exposure during the last third of gestation, including the BGS, decreases brain weight that persists into postnatal life, which is associated with growth restriction of the cerebral cortex. However, this prenatal ethanol exposure regimen, including the BGS, does not increase spontaneous locomotor activity in contrast to the persistent hyperactivity that occurs after chronic ethanol exposure throughout gestation. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Prenatal ethanol exposure; Brain growth spurt; Cerebral cortex; Hippocampus; Cerebellum; Spontaneous locomotor activity; Guinea pig
1. Introduction Consumption of ethanol during pregnancy can produce a broad spectrum of toxic effects on the developing fetus, including teratogenicity, spontaneous abortion, and stillbirth [3]. The teratogenic effects of ethanol in the human can present as the fetal alcohol syndrome (FAS) [14]. The most debilitating and permanent consequence of ethanol teratogenesis is the central nervous system (CNS) dysfunction, which can manifest as intellectual, neurological, and behavioural abnormalities. Experimental animal studies have demonstrated that the cerebral cortex, hippocampus, and cerebellum are among the brain target sites of ethanol CNS teratogenesis [24,34]. Considerable advances have been made in the last decade in our understanding of FAS, but
* Corresponding author. Tel.: +1-613-533-6114; fax: +1-613-5336412. E-mail address: [email protected] (J.F. Brien).
the mechanism(s) of ethanol CNS teratogenesis remain(s) to be elucidated [4,34]. The development of the CNS in the human and other mammalian species can be divided into distinct stages, but with considerable overlap of the ontogenic events that occur [24]. Exposure to ethanol during different stages of gestation can produce different deleterious intellectual, neurological, and/or behavioural outcomes, thereby reflecting temporal vulnerability during development. The developing brain may be especially sensitive to the teratogenic effects of ethanol during the brain growth spurt (BGS), which is characterized by a rapid increase in brain weight, proliferation of astroglial and oligodendroglial cells, neuronal axonal elongation, dendritic arborization, and synaptogenesis [24]. In the human, the BGS is a perinatal event that begins in the third trimester and continues into early postnatal life [19]. It has been suggested that cessation of maternal consumption of ethanol before the beginning of the third trimester can limit the severity or prevent the occurrence of some of ethanol’s CNS teratogenic effects [16,17].
0892-0362/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 2 - 0 3 6 2 ( 0 1 ) 0 0 1 5 0 - 7
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Another important factor influencing the CNS teratogenic effects of ethanol for exposure during distinct periods of development is the fact that not all brain regions develop at the same time, and thus, depending on the developmental stage during which ethanol exposure occurs, some brain regions may be more affected than others [24]. In addition, within a given brain region, different neuronal cell populations have different temporal patterns of development that may influence their relative vulnerability to the teratogenic effects of ethanol [24]. When using animal models to study temporal vulnerability to ethanol exposure, one must take into account the timing of the various stages of brain development, such as the BGS, in relation to birth. In the rat, the BGS occurs primarily after birth, with the majority of the BGS occurring during the first 2 weeks of postnatal life [35]. Thus, exposure to ethanol during the BGS in the rat requires administration of ethanol to the neonate. The objective of this study was to characterize, in the guinea pig, ethanol CNS teratogenicity for prenatal ethanol exposure during the last third of gestation, including the BGS. The following hypothesis was tested: prenatal ethanol exposure during the last third of gestation, including the BGS, in the guinea pig produces neurobehavioural teratogenicity, manifesting as brain growth restriction and hyperactivity. The guinea pig was selected as the experimental animal because of its extensive prenatal brain development including the BGS, the majority of which occurs between gestational day (GD) 45 and 55 (term, about GD 68) [18]. Therefore, in the guinea pig, maternal administration of ethanol during the last third of gestation results in offspring being exposed to ethanol in utero during the BGS, an apparent critical period of vulnerability. People with FAS have been exposed to ethanol during prenatal life, as a consequence of maternal ingestion of ethanol. Thus, administration of ethanol to the pregnant guinea pig would appear to model more closely the human situation, than is the case for the rat.
2. Methods 2.1. Experimental animals Nulliparous female Dunkin – Hartley strain guinea pigs (500 –600 g body weight) were bred with male Dunkin – Hartley strain guinea pigs using an established procedure [22]. GD 0 was defined as the last day of full vaginalmembrane opening. On GD 1, pregnant animals were housed individually in cages in a room with a 12-h light/ 12-h dark cycle with lights on at 0700 h and ambient room temperature of 23C. Vaginal-membrane status and general health of the pregnant animals were monitored, and body weight was measured daily throughout gestation. All guinea pigs were cared for according to the principles and guidelines of the Canadian Council on Animal Care. The experi-
mental protocol was approved by the Queen’s University Animal Care Committee. 2.2. Animal treatment regimens and maternal blood ethanol concentration Two investigations were conducted, in which offspring were studied prenatally at GD 63 or postnatally at PD 10. In the GD 63 study, six pregnant guinea pigs were studied for each of the ethanol, isocaloric-sucrose/pair-fed and water treatment groups; in the PD 10 study, four pregnant guinea pigs were studied for each treatment group. A preliminary study demonstrated that oral administration of 4 g ethanol/kg maternal body weight per day starting on GD 45 resulted in maternal death in 5 of 11 pregnant guinea pigs. In view of this 45% incidence of maternal lethality, the ethanol dosage regimen was modified to provide maternal exposure to a lower daily dose of ethanol (2 g ethanol/kg maternal body weight per day) starting on GD 43, for the first 2 days of treatment in order to allow the pregnant animal to develop tolerance to the maternal toxicity of the 4 g ethanol/kg maternal body weight per day regimen, which began on GD 45. Each pregnant animal was randomly assigned to one of the following oral treatment regimens. The ethanol regimen was administered as 2 g ethanol/kg maternal body weight per day on GD 43 and/or 44 and then as 4 g ethanol/kg maternal body weight per day from GD 45 to 62 in the form of an aqueous ethanol solution (30% v/v in tap water) with ad libitum access to pellet food (Purina Guinea Pig Chow 50251) and water. The 4 g ethanol/kg maternal body weight per day regimen was selected for study because it has been shown to produce persistent neurobehavioural changes in offspring, with minimal embryonic/fetal or maternal lethality, when given throughout gestation [2,12]. For the isocaloric-sucrose/pair-feeding regimen, each pregnant animal was paired to an ethanoltreated guinea pig, received sucrose (42% w/v in tap water, isocaloric and isovolumetric to the daily dose of ethanol) and was given food in an amount equivalent to that consumed by the ethanol-treated animal, with free access to water. Virtually all the food provided on each day of the treatment period was consumed by the pregnant animal. For the water regimen, water (isovolumetric to the daily dose of ethanol) was administered with ad libitum access to food and water. Each animal received the daily treatment by oral intubation into the mouth as two equally divided doses 2 h apart, with the first dose given between 1000 and 1200 h and the second dose given 2 h later. Each animal was treated from GD 43 to 62 (term, about GD 68) to include the BGS. Thereafter, each pregnant animal had ad libitum access to food and water. On GD 57 or 58, maternal blood was taken from an ear blood vessel at 1 h after the second divided dose of ethanol, isocaloric-sucrose, or water for the determination of ethanol concentration by an established gas-liquid
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chromatographic procedure [32]. The 200-ml blood sample was collected from each pregnant guinea pig in the study over a maximum time period of 5 min, in which the animal was restrained only during the blood vessel puncture. The time of blood sampling was selected in view of a previous pharmacokinetic study [13] of ethanol disposition in the maternal-fetal unit, in which the apparent maximum blood ethanol concentration (BEC) occurred at 1 h after the last divided dose of 4 g ethanol/kg maternal body weight. Blood sampling was performed on GD 57 or 58 in order to compare the ethanol concentration data with those determined in previous studies, in which ethanol was administered throughout gestation. 2.3. Pregnancy outcome At birth (PD 0), each maternal guinea pig and its litter were moved from the cage housing, used up to and including parturition, to a large plastic bin with wood chip bedding. The litter size and number of live offspring were recorded, and the presence of gross dysmorphology was assessed. On PD 1, the body weight and gender of the offspring of each litter were determined. The offspring were monitored daily for body weight and general health. Perinatal death was defined as death that occurred at parturition or during the first 10 days of postnatal life. 2.4. Measurement of spontaneous locomotor activity On PD 10, the cumulative spontaneous locomotor activity of the individual offspring from each litter was measured in an open-field apparatus using an Opto-Varimex1 monitor (Columbus Instruments; Columbus, OH). The apparatus (42 42 21 cm high) was equipped with blackened walls, a clear plastic top, and two arrays of infrared beams that were set at 3 cm above the floor to measure horizontal spontaneous locomotor activity (ambulatory, exploratory movement) and at 8 cm above the floor to measure vertical spontaneous locomotor activity (climbing, rearing). The cumulative number of infrared-beam breaks in each of the horizontal and vertical planes were recorded at 10-min intervals for a 60-min period. The testing was performed in a room with ambient temperature of 23C and standard fluorescent lighting, in which background noise was minimized. 2.5. Offspring body, brain, and brain region weights On GD 63, individual pregnant guinea pigs from each of the three treatment groups were euthanized by halothane anesthesia followed by decapitation. The litter of each pregnant animal was delivered by cesarean section, and each fetus was decapitated. On PD 10, randomly selected offspring from each litter were weighed, and euthanized by halothane anesthesia followed by decapitation. The offspring’s brain was excised and weighed. The
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hippocampus, cerebral cortex, and cerebellum were dissected and weighed. 2.6. Data analysis The data are presented as group means ± S.E.M. of the average littermate values for the individual litters. There was no heterogeneity of variance for the data of the three treatment groups, as determined by Bartlett’s test. Therefore, parametric statistical analysis was conducted using analysis of variance (ANOVA). For the weight data, oneway, randomized-design ANOVA was conducted. If a treatment effect was indicated by a statistically significant F statistic ( P < 0.05), then Newman-Keuls post hoc test was conducted to determine which treatment groups were statistically different. Spontaneous locomotor activity data were analyzed using two-way ANOVA with respect to treatment and time.
3. Results 3.1. Maternal BEC, food intake, and body weight The maternal BEC of the pregnant guinea pigs in the ethanol treatment group at 1 h after the second divided dose on GD 57 or 58 was 340 ± 76 mg/dl (n = 8). There was no measurable ethanol in the blood of the pregnant guinea pigs that received isocaloric-sucrose/pair-feeding or water treatment. The average daily food intake and maternal body weight data of the pregnant guinea pigs in the three experimental groups for the GD 43 to 62 treatment period are presented in Fig. 1 for the study involving GD 63 offspring and in Fig. 2 for the study involving PD 10 offspring. For each study, the average daily food intake of the pregnant animals was decreased in the ethanol treatment group compared with the water treatment group ( P < 0.05). However, the maternal body weight during and after the treatment period was not different among the ethanol, isocaloric-sucrose/pair-fed and water treatment groups. 3.2. Pregnancy outcome The pregnancy outcome data for the three treatment groups of each of the two studies at GD 63 and PD 10 are presented in Tables 1 and 2, respectively. There was no maternal death for the ethanol, isocaloric-sucrose/pairfed, and water treatment groups of the two studies. There was one incident of spontaneous abortion in the isocaloric-sucrose/pair-fed pregnant guinea pigs of the study at GD 63, and two incidents of spontaneous abortion in the ethanol-treated pregnant guinea pigs of the study at PD 10. There was no perinatal death for the three treatment groups of the two studies. There was no difference in litter size, or percentage of male and female littermates
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neous locomotor activity during the 60-min period ( P < 0.05). 3.4. Offspring body, brain, and brain region weights There was no gender difference in the offspring body, brain, cerebral cortical, hippocampal, or cerebellar weight data for any of the three treatment groups at GD 63 or PD 10 (data not shown). Consequently, the data of the male and female offspring in each treatment group were combined. The body, brain, cerebral cortical, hippocampal, and cerebellar weight data for GD 63 offspring of the three treatment groups are presented in Fig. 4. There was no difference in body weight among the three treatment groups. Brain, cerebral cortical, hippocampal, and cerebellar weights were decreased by 17.3%, 19.7%, 13.6%, and 13.4%, respectively, for the ethanol group compared with the isocaloric-sucrose/pair-fed control group ( P < 0.05), and were decreased by 21.1%, 21.1%, 23.3%, and 20.9%,
Fig. 1. Average daily food intake during treatment and maternal body weight of pregnant guinea pigs that received oral administration of ethanol (n = 6), isocaloric-sucrose/pair-feeding (n = 6) or water (n = 6) during the last third of gestation, including the BGS (GD 43 to GD 62; term, about GD 68), for the GD 63 study. The data are presented as group means ± S.E.M. Treatment period is indicated by the shaded box. Groups means with different letters are statistically different from each other ( P < 0.05).
among the three treatment groups at GD 63. There was no difference in length of gestation, litter size, average littermate birth weight, or percentage of male and female littermates among the three treatment groups at PD 10. 3.3. Spontaneous locomotor activity There was no gender difference in the spontaneous locomotor activity data of PD 10 offspring for any of the three treatment groups (data not shown), and the data of the male and female offspring in each treatment group were combined. The cumulative horizontal and vertical spontaneous locomotor activity data for PD 10 male and female offspring of the three treatment groups for the 60min observation period are presented in Fig. 3. Based on statistical analysis of the four litters in each treatment group, there was no apparent treatment effect for horizontal or vertical spontaneous locomotor activity among the ethanol, isocaloric-sucrose/pair-fed and water groups, but there was a time effect of cumulative horizontal sponta-
Fig. 2. Average daily food intake during treatment and material body weight of pregnant guinea pigs that received oral administration of ethanol (n = 4), isocaloric-sucrose/pair feeding (n = 4) or water (n = 4) during the last third of gestation, including the BGS (GD 43 to GD 62; term, about GD 68), for the PD 10 study. The data are presented as group means ± S.E.M. Treatment period is indicated by the shaded box. Groups means with different letters are statistically different from each other ( P < 0.05).
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Table 1 Effects of maternal administration of ethanol, isocaloric-sucrose/pair-feeding, or water during the last third of gestation, including the BGS, on pregnant outcome of the guinea pigs whose offspring were studied at GD 63 Pregnancy outcome variable
Ethanol (6)
Treatment sucrose (7)
Water (6)
Maternal death Spontaneous abortion Perinatal death Litter size Male littermates (%) Female littermates (%)
0 0 0 3.7 ± 0.8 (6) 49.7 ± 17.7 (6) 50.3 ± 17.7 (6)
0 1 0 3.8 ± 0.9 (6) 62.2 ± 26.5 (6) 37.8 ± 26.5 (6)
0 0 0 3.3 ± 0.5 (6) 54.2 ± 18.1 (6) 45.8 ± 18.1 (6)
The number of pregnant guinea pigs or litters is reported in parentheses. The data for maternal death, spontaneous abortion, and perinatal death are reported as the number of occurrences. The other data are reported as group means ± S.E.M. of average littermate values for the individual litters.
respectively, for the ethanol group compared with the water control group ( P < 0.05). However, there was no difference in the brain region weight/brain weight ratio data among the ethanol, isocaloric-sucrose/pair-fed and water treatment groups, respectively: cerebral cortical weight/brain weight ratio, 0.578 ± 0.008, 0.594 ± 0.008, and 0.577 ± 0.007, respectively; hippocampal weight/ brain weight ratio, 0.088 ± 0.005, 0.084 ± 0.002, and 0.091 ± 0.003, respectively; and cerebellar weight/brain weight ratio, 0.107 ± 0.004, 0.103 ± 0.002, and 0.107 ± 0.003, respectively. The body, brain, cerebral cortical, hippocampal, and cerebellar weight data for PD 10 male and female offspring of the three treatment groups are presented in Fig. 5. There was no difference in body, hippocampal, or cerebellar weight among the three treatment groups. Brain and cerebral cortical weights were decreased by 11.6% and 17.1%, respectively, for the ethanol group compared with the isocaloric-sucrose/pair-fed group ( P < 0.05), and were decreased by 15.2% and 16.3%, respectively, for the ethanol group compared with the water control group ( P < 0.05). However, there was no difference in the brain region weight/ brain weight ratio data among the ethanol, isocaloricsucrose/pair-fed, and water treatment groups, respectively; cerebral cortical weight/brain weight ratio, 0.556 ± 0.007, 0.587 ± 0.009, and 0.566 ± 0.010, respectively; hippocampal weight/brain weight ratio, 0.078 ± 0.002, 0.074 ± 0.001,
and 0.072 ± 0.001, respectively; and cerebellar weight/ brain weight ratio, 0.117 ± 0.003, 0.113 ± 0.003, and 0.115 ± 0.003, respectively.
4. Discussion Researchers have been successful in using animal models to investigate the teratogenic effects of ethanol on the developing CNS [21]. In spite of the extensive knowledge of FAS [4,29,34], several important questions regarding the factors that influence the severity of prenatal ethanol exposure-induced CNS damage remain unanswered. One of the most interesting of these questions, from both clinical and experimental points of view, focuses on critical periods during development when the brain may be particularly vulnerable to ethanol exposure, including the BGS. The guinea pig as an experimental animal allows for the examination of both the direct effects (i.e., target fetal site) and indirect effects (i.e., maternal, placental, or other fetal sites) of maternal ethanol administration on fetal brain development during the BGS, which occurs prenatally in this animal species [29]. In the rat, the BGS occurs primarily postnatally [24,28]. Thus, any study attempting to investigate the effects of ethanol on the brain during the BGS in the rat must involve postnatal exposure [28]. Also,
Table 2 Effects of maternal administration of ethanol, isocaloric-sucrose/pair feeding, or water during the last third of gestation, including the BGS, on pregnancy outcome of the guinea pigs whose offspring were studied at PD 10 Pregnancy outcome variable
Ethanol (6)
Treatment sucrose (4)
Water (5)
Maternal death Spontaneous abortion Perinatal death Length of gestation Litter size Average littermate birth weight (g) Male littermates (%) Female littermates (%)
0 2 0 68.3 ± 1.0 (4) 3.4 ± 0.6 (4) 96.2 ± 7.5 (4) 45.8 ± 41.7 (4) 54.2 ± 41.7 (4)
0 1 0 67.8 ± 1.0 (4) 3.0 ± 1.4 (4) 102.9 ± 20.8 (4) 20.8 ± 14.4 (4) 79.2 ± 14.4 (4)
0 0 0 68.6 ± 1.5 (5) 2.4 ± 0.9 (5) 110.7 ± 12.3 (5) 56.7 ± 27.9 (5) 43.3 ± 27.9 (5)
The number of pregnant guinea pigs or litters is reported in parentheses. The data for maternal death, spontaneous abortion, and perinatal death are reported as the number of occurrences. The other data are reported as group means ± S.E.M. of average littermate values for the individual litters.
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Fig. 3. Cumulative horizontal and vertical spontaneous locomotor activity data for a 60-min period of observation of PD 10 offspring of pregnant guinea pigs that received oral administration of ethanol (n = 4 litters), isocaloric-sucrose/pair-feeding (n = 4 litters), or water (n = 4 litters) during the last third of gestation, including the BGS (GD 43 to GD 62; term, about GD 68). The data are presented as group means ± S.E.M. of average littermate values for the individual litters. There was statistically significant effect of time (across the 60-min observation period) for cumulative horizontal activity, F(5,54) = 13.78, P < 0.05.
the potential contribution of ethanol-induced changes in maternal or placental function on offspring brain development cannot be examined in the postnatal rat model [29]. The disposition of ethanol in the maternal-fetal unit in the guinea pig is similar to that for the human situation [8,11,13]. In view of the fact that the neonatal rat has a lower capacity for hepatic biotransformation of ethanol than the adult [31], for a given dose of ethanol normalized to body weight, it would experience a higher BEC for a longer time period than the fetal guinea pig exposed to ethanol in utero, because the predominant route of ethanol
elimination from the maternal-fetal unit is via maternal hepatic biotransformation [8,13]. Thus, in order to clarify that the third trimester is a critical period of brain development for the teratogenic effects of ethanol, more investigation should be conducted using a species that possesses prenatal brain growth characteristics similar to those of the human (e.g., the guinea pig) [33]. In the present study in the guinea pig with maternal ethanol administration restricted to the developmental period from GD 43 to 62, which includes the majority of the BGS, the maternal BEC on GD 57 or 58 at 1 h after
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Fig. 4. Body, brain cerebral cortical, hippocampal, and cerebellar weights for GD 63 offspring of pregnant guinea pigs that received oral administration of ethanol (n = 6 litters), isocaloric-sucrose/pair-feeding (n = 6 litters) during the last third of gestation, including the BGS (GD 43 to GD 62; term, about GD 68). The data are presented as group means ± S.E.M. of average littermate values for the individual litters. Group means with different letters are statistically different from each other ( P < 0.05): brain weight, F(2,15) = 32.09, P < 0.05; cerebral cortical weight, F(2,15) = 17.00, P < 0.05; hippocampal weight, F(2,15) = 11.37, P < 0.05; cerebral weight, F(2,15) = 9.25, P < 0.05.
the daily ethanol dose was about 1.5- to 2-fold higher than the maternal BEC for our previous studies of ethanol teratogenicity, in which the same 4 g ethanol/kg maternal
body weight per day regimen was administered chronically from GD 2 to 67 [1,2,6,9,12,23,25,26]. This difference appears to result from altered pharmacokinetics of ethanol
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Fig. 5. Body, brain, cerebral cortical, hippocampal, and cerebellar weights for PD 10 offspring of pregnant guinea pigs that received oral administration of ethanol (n = 4 litters), isocaloric-sucrose/pair-feeding (n = 4 litters) or water (n = 4 litters) during the last third of gestation, including the BGS (GD 43 to GD 62; term, about GD 68) The data are presented as group means ± S.E.M. of average littermate values for the individual litters. Group means with different letters are statistically different from each other ( P < 0.05): brain weight, F(2,9) = 22.5, P < 0.05; cerebral cortical weight, F(2,9) = 19.98, P < 0.05.
in the maternal-fetal unit due to greater gastrointestinal absorption of ethanol and/or slower elimination of ethanol, via maternal hepatic biotransformation, for the ethanol dosage regimen during the last third of gestation, including
the BGS. It is noteworthy that chronic ethanol ingestion by the adult can induce cytochrome P450 (CYP) 2E1 in the liver and that CYP2E1 catalyzes the hepatic oxidation of ethanol and, hence, the elimination of ethanol at high
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substrate concentration when the alcohol dehydrogenase pathway is saturated [27]. It is reasonable to speculate that the higher maternal BEC in the pregnant guinea pigs that received ethanol only during the last third of gestation, including the BGS, is due to normal rate of hepatic biotransformation of ethanol for this stage of gestation (GD 57 or 58) with no, or minimal, induction of the CYP2E1 metabolic pathway. This is contrast to the situation for chronic ethanol exposure up to this stage of pregnancy (GD 57 or 58) that would induce CYP2E1 and increase maternal hepatic biotransformation of ethanol and the rate of ethanol elimination from the maternal-fetal unit. In the present study, offspring in the ethanol treatment group studied at GD 63 had decreased brain, cerebral cortical, hippocampal, and cerebellar weights compared with the isocaloric-sucrose/pair-fed and water treatment groups. However, offspring in the ethanol treatment group studied at PD 10 only had decreased brain and cerebral cortical weights compared with the isocaloric-sucrose/pairfed and water treatment groups, with no change in hippocampal or cerebellar weight. The data demonstrate that, in the guinea pig, prenatal ethanol exposure during the last third of gestation, including the BGS, decreases brain weight that persists into postnatal life, which is associated with a persistent decrease in cerebral cortical weight. No change was observed in body weight of GD 63 or PD 10 offspring of the ethanol treatment group compared with the isocaloric-sucrose/pair-fed and water treatment groups, indicating that there was selective brain growth restriction. The ethanol-induced decrease in brain and cerebral cortical weights in the young postnatal guinea pig suggests that, although the brain goes through a period of rapid growth during the last third of gestation, each brain region has its own temporal growth spurt and, hence, its own critical period of vulnerability. The data of the present study should not be interpreted to suggest that prenatal ethanol exposure during the last third of gestation, including the BGS, does not alter physiological or biochemical function and/or structure of different neuronal cell types in the hippocampus and cerebellum in postnatal life. Clearly, more research is required before it can be stated that the hippocampus and cerebellum are not vulnerable to the teratogenic effects of prenatal ethanol exposure during the BGS. In view of the fact that the hippocampus of the guinea pig is partially formed by GD 45 (unpublished observation), it is conceivable that the critical period of vulnerability of the hippocampus to prenatal ethanol exposure is earlier in gestation than the general BGS. In addition to microencephaly, hyperactivity is a key behavioural manifestation of FAS in the human and ethanol CNS teratogenicity in experimental animals [21]. Several studies in our laboratory have demonstrated that, in the guinea pig, chronic prenatal ethanol exposure throughout gestation results in a persistent increase in spontaneous locomotor activity and decrease in brain weight in the
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offspring of the ethanol treatment group compared with the isocaloric-sucrose/pair-fed and water treatment groups [2,7,9,23]. In contrast, in our study in which prenatal ethanol exposure was restricted to a time period during the last third of gestation, including the BGS, there was decreased brain weight, but no apparent difference in cumulative horizontal or vertical spontaneous locomotor activity of the offspring at PD 10 among the three treatment groups. Hence, there would appear to be a dissociation between the neurobehavioural deficits produced by chronic ethanol exposure throughout gestation, manifesting as microencephaly and hyperactivity, and the apparent lack of behavioural dysfunction in the presence of microencephaly for ethanol exposure during the BGS. It is conceivable that more sophisticated behavioural assessment, such as the Morris water maze test, which the guinea pig will perform [20], would reveal neurobehavioural dysfunction for ethanol exposure during the BGS. Experiments currently are being conducted to test this concept. Experimental animal studies have shown that there are similarities between the neurobehavioural dysfunction, produced by chronic prenatal ethanol exposure, and by postnatal hippocampal lesioning, such as hyperactivity [30]. The mechanism by which chronic prenatal ethanol exposure produces hyperactivity in the human and experimental animals, including the guinea pig, is not clearly understood. For brain ischemia in the adult gerbil, increased spontaneous locomotor activity is associated with a selective loss of hippocampal CA1 pyramidal cells [5,15]. Similarly, in the young postnatal guinea pig (PD 10) exposed to ethanol prenatally throughout gestation, there is a 30% loss of CA1 pyramidal cells and decreased hippocampal weight, both of which are temporally associated with the hyperactivity [23]. In our study, in which prenatal ethanol exposure was restricted to a time period during the last third of gestation, including the BGS, there was no effect on spontaneous locomotor activity or hippocampal weight, and in a preliminary study, there was no apparent effect on the number of hippocampal CA1 or CA3 pyramidal cells, or dentate gyrus granule cells in PD 10 guinea pig offspring [10]. These findings are consistent with the idea that CA1 pyramidal cell injury as a consequence of prenatal ethanol exposure may be involved in the increased spontaneous locomotor activity. In conclusion, our data demonstrate that, in the guinea pig, prenatal ethanol exposure during the last third of gestation, including the BGS, decreases brain weight that persists into postnatal life and is associated with growth restriction of the cerebral cortex, but does not increase spontaneous locomotor activity. These effects are in contrast to the persistent hyperactivity and decreased brain weight that occur after chronic prenatal ethanol exposure throughout gestation. In order to characterize fully the third trimester, and the BGS in particular, as a critical period of vulnerability for the CNS teratogenic effects of ethanol, more investigation is required using an experimental ani-
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mal, such as the guinea pig, that possesses prenatal and postnatal brain growth characteristics similar to those of the human [33].
Acknowledgments This research was supported by an operating grant (MT8073) from the Canadian Institutes of Health Research. MLB is the recipient of an Eldon Boyd Fellowship, Department of Pharmacology and Toxicology, Queen’s University.
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